This invention relates to substances used to enhance or induce an immune response to an antigen. In particular, it is directed to the use of substances normally found in the body for such purposes.
Immunization is a critical component of many industrial and therapeutic processes. The capability to raise high titer, high specificity antisera against a selected antigen is important in the manufacture of antibodies for in vitro and in vivo use and for ensuring successful vaccinations. Vaccination is becoming significant outside its well-known role in protecting against infectious diseases. For example, vaccination against self or syngeneic tumors is a developing technology1, as is the use of vaccination to control physiological processes such as reproduction and growth by inducing autoimmune responses that antagonize or agonize biological effector molecules in vivo. In all of these new approaches it is important to induce a vigorous immune response to the target antigen, either humoral or cell mediated. This heretofore has been accomplished by administering an adjuvant in connection with the antigen, and in some cases by complexing the antigen with a carrier. Adjuvants and carriers are substances that in themselves share no immune epitopes with the target antigen but which stimulate the immune response to the target antigen. Freund""s adjuvant, a mineral oil emulsion, commonly has been used for this purpose, as have a variety of toxic microbial substances such as mycobacterial extracts59, 60. Carriers often act as adjuvants as well, but are generally distinguished from adjuvants in that carriers comprise water insoluble macromolecular particulate structures which aggregate the antigen. Typical carriers include aluminum hydroxide, latex particles, bentonite and liposomes.
Many diverse materials have been demonstrated to possess adjuvant activity, and their mechanisms of action are nearly as varied, as is apparent from several reviews on the subject61,62,63,64. Adjuvant effects have been attributed to antigen aggregation, antigen depot formation, altered lymphocyte recirculation, stimulation of T lymphocytes, mitogenic effects on B lymphocytes, activation of phospholipase A, inhibition of prostaglandin synthesis, cell membrane alterations, localization of antigens in thymus-dependent areas of lymph nodes, modified antigen processing by macrophages, and stimulation of macrophage replication and activation65. It is clear that the biology of adjuvants is quite complex and that a number of hypotheses exist for the mode of action of immune adjuvants.
Monokines such as interleukin-1 have-been implicated as mediators in adjuvant effects. The adjuvant effect of IL-1 has been attributed to its activity as a lymphocyte growth factor65,66,47. Certain synthetic adjuvants also are known to be able to induce interferon synthesis65 and coadministration of antigen and xcex3-interferon to mice is known to potentiate immune response to the antigen9.
Potentially undesirable effects of the heretofore available adjuvants and adjuvant formulations are well known. A list of side effects of adjuvants includes: (1) sensitization to tuberculin or any other antigen used in screening tests for infections; (2) presence in food animals of materials that cannot safely be ingested by humans; (3) inflammatory, granulomatous, necrotizing, or other unacceptable reactions at injection sites most notably as occurs with Freund""s complete adjuvant; (4) pyrogenicity; (5) central nervous system effects and untoward behavioral effects; (6) impairment of growth; (7) arthritis; (8) increased vascular permeability and inflammatory reactions in the eye; (9) induction of undesired autoimmune responses and (10) immune suppression for adjuvant epitopes.
Whether the induction of autoimmune responses is undesirable depends upon the therapeutic objective. For example, the induction of autoimmune responses such as allergic encephalomyelitis in a small minority of subjects would be a very undesirable property of an adjuvant for human use. In contrast, the generation of an autoimmune response is frequently the objective in the veterinary field, e.g., suppressing fertility in animals by inducing autoantibodies against luteinizing hormone-releasing hormone, increasing fertility by vaccinating against endogenous inhibin, or increasing growth by eliciting autoantibodies against somatostatin, and is also desirable in inducing an immune response to host tumors.
Vaccination adjuvants are needed which are capable of use so as to be free of the undesirable side effects noted, above. Accordingly, it is an object of the invention herein to enhance the titer and duration of the mammalian immune response, both humoral and cellular, without toxic reactions. This and other objects of the invention will be apparent from the specification as a whole.
Monocytes and lymphocytes are known to produce the cytokines TNF-xcex18 and TNF-xcex2 (previously called lymphotoxin)2,3, respectively. Their complete primary structures have been determined and the cDNAs of both TNF-xcex24 and TNF-xcex15-7,10 have been cloned by recombinant DNA methods and expressed in E. coli. 
In vivo and in vitro studies using the pure TNFs have shown that both TNF-xcex1 and TNF-xcex2 possess the unique ability to kill neoplastic tissue selectively, while sparing most normal cells. In addition to their antitumor activity, these proteins mediate a diverse array of biological responses in vitro. Although their true in vivo significance is still unknown, the biologic studies strongly suggest that TNF-xcex1 and TNF-xcex2 play an important role in immunomodulatory and inflammoatory responses.
TNF-xcex1 and TNF-xcex2 differ significantly in their physical and chemical properties. TNF-xcex1 is a 157 residue polypeptide with a molecular weight of 17,0008 by SDS-PAGE. Under the same conditions, two different forms of TNF-xcex2, with molecular weights of about 20,000 (148 residues) and 25,000 (171 residues), have been found2,3. The 20 kD species is a proteolytic cleavage product of the 25 kD form.2,3 The molecular weight of TNFs under non-denaturing conditions are very different. Purified human TNF-xcex1 has a native molecular weight of 45,0008, whereas TNF-xcex2 elutes at a position corresponding to a molecular weight of 60-70,0002,3 during gel filtration. The isoelectric points (pI) of these cyto-toxic factors have been reported to be in the range of 4.5-6.5. TNF-xcex2 has a pI of 5.8, and 5.3 is the pI determined for TNF-xcex1.
The amino acid sequence of human TNF-xcex1 as determined from the protein6,7,11 or predicted from the nucleotide sequence6,7,10,11 has been described. Some variations in the protein sequences at the amino terminal end have been observed. The N-terminal protein sequence of the natural human TNF-xcex1 purified from HL-60 cells obtained by Wang et al.,7 has two discrepancies with the sequence reported earlier8 and that predicted from the cloned cDNA sequence5,7. Two out of the three serines in positions 3-5 of the mature protein are missing, and the His-Val sequence in position 15 and 16 has been replaced by Val-Ser-Val-Ser. The reason for this discrepancy is not clear. Two groups6,11 have reported the N-terminal sequence of recombinant human TNF-xcex1 expressed and purified in E. coli in which Val-Arg from position 1 and 2 of the natural protein sequence, respectively, are missing, even though the nucleotide codons for these amino acids are present at the corresponding positions in both the genomic6 and cDNA11 sequences. These investigations assumed that the N-terminal sequence of human TNF-xcex1 was Ser-Ser-Ser-Arg-. . . based on an analogy with the sequence of rabbit TNF-xcex1 purified from serum.
TNF-xcex1 has been extensively studied and has been found to exert a variety of effects on normal cells. One major indication of an effect of TNF-xcex1 on normal tissue stems from studies on cachectin12,13,14, a macrophage secreted factor that inhibited the synthesis of lipoprotein lipase in the mouse adipocyte cell line 3T3-L1. Cachectin has been suggested to be the agent responsible for causing cachexia during certain chronic host infections and malignancies.14,15 One of the salient features of cachexia is the loss of body weight, even with adequate food consumption. The purification of cachectin and its partial structure determination revealed that this protein was identical to TNF-xcex1.12,13 These studies have prompted the suggestion that TNF-xcex1 is the agent responsible for cachexia during chronic host infection.14,15 However, recent studies indicate that this activity is not unique to TNF-xcex1, since other cytokines including IL-116, IFNs17,18, and TNF-xcex218 can also suppress lipoprotein lipase activity in 3T3-L1 adipocytes.
Tumor necrosis serum (TNS) and partially purified preparations of TNF-xcex1 have been reported to protect animals against bacterial and parasitic infections. C3H/HeJ mice challenged with Klebsiella pneumoniae or Listeria monocytogenes showed increased survival rates following TNS injection compared to untreated controls.19 TNF-xcex1 also appears to have a potent cytotoxic effect on the malarial parasites Plasmodium falciparum20, Plasmodium yoelii and Plasmodium berghei.21 Recently recombinant TNF-xcex1 was shown to be similar to eosinophil cytotoxicity enhancing factor and it potentiated eosinophil cytotoxicity against Schistosoma mansoni larvae.22 
Several investigators have found that rTNF-xcex1 exhibits direct antiviral activity similar to interferons. TNF-xcex1 protected HEP-2 cells against VSV infection24 and this effect was not blocked by anti-IFN antibodies.24 Similarly, TNF-xcex1 and TNF-xcex2 were shown to directly induce resistance to infection by both RNA viruses (EMCV and VSV) and DNA viruses (Ad-2 and HSV-2) in diverse cell types.25 The antiviral effect of TNFs was not IFN-mediated since it was not abolished by anti-IFN-xcex1, -xcex2, or -xcex3 antibodies, there were no detectable levels of IFNs in the cell culture fluids, and no IFN mRNA was found in the cells. In addition to inducing the antiviral state, TNFs were also able to selectively kill virus-infected cells. Both the antiviral activity and the virus-induced cytotoxicity of TNFs were synergistically enhanced by IFNs.25 Furthermore, viruses as well as the polymer poly(I):poly(C) could induce the production of TNF-xcex1 in HL-60 cells and TNF-xcex2, in RPMI 1788 cells.25 
A role for TNF in mediating inflammatory responses has been implicated by its effects on neutrophil functions. It has been reported26 that pretreatment of PMN with purified TNF-xcex1 and TNF-xcex2, (free of detectable LPS contamination) induces a significant increase in their ability to phagocytose fluorescein-conjugated latex beads as well as an enhancement of PMN-mediated antibody dependent cellular cytotoxicity (ADCC) against chicken erythrocytes. More recently other investigators27 have found significant increases in phagocytosis of unopsonized zymosan particles, degranulation, and respiratory burst activity by TNF-xcex1 treated PMN. Interestingly, these effects were inhibited by monoclonal antibodies against the C3bi receptor/adherence glycoprotein CD11.28 TNF-xcex1 has been shown29 to increase the expression of this protein on neutrophils resulting in their enhanced adherence to the endothelium.
In addition to its effects on PMN,TNF-xcex1 appears to have direct effects on endothelial cells which play a major role in inflammation and tissue injury. TNF-xcex1 induced the release of IL-1 from endothelial cells30 and induces neutrophil adherence to endothelial cells29,31 via the CDW18 neutrophil membrane protein complex (also called the C3bi receptor/adherence glycoprotein mentioned earlier). Further evidence that the endothelium is a major site of TNF action in vivo comes from studies on effects of TNF-xcex1 on the hemostatic properties of endothelial cells33 in culture. Incubation with rTNF-xcex1 causes changes in the production of two activities:33 induction of tissue factor, a procoagulant cofactor protein34,35, and inhibition of formation of activated protein C, an anticoagulant cofactor protein36. Extensive changes in the morphology of human vascular endothelial cells in confluent primary culture treated with TNF-xcex1 have also been reported37.
A possible role for TNF-xcex1 and TNF-xcex2, in granulocyte-macrophage differentiation has been suggested from recent observations by two groups of investigators who have found that TNF-xcex141 and TNF-xcex242 suppress colony formation by bone-marrow derived hematopoietic progenitor cells. Low doses (0.05-100 U/ml) of rTNF-xcex141 as well as TNF-(Luk II)43 inhibited granulocyte-macrophage differentiation by both late (CFU-GM, day 7) and early (DFU-GM, day 14) precursor cells. The effect was rapid, since pulsing the bone marrow cells for only one hour with TNF-xcex1 was sufficient to cause suppression. Inhibition of colony formation by erythroid (BFU-E) as well as multi-potential (CFU-GEMM) progenitor cells was also observed41. All of these effects could be seen in non-adherent bone marrow cells substantially depleted (down to 2 percent) of monocytes and T lymphocytes41. The inhibitory effect of TNF-xcex2 on CFU-GM was observed in the presence of IFN-xcex342.
A number of intracellular activities have been reported. They include stimulation of IL-1 and PGE2 production in resting macrophages44, induction of class I (but not the immunologically more significant class II antigens) protein antigens of the major histocompatibility complex32, induction of synthesis of collagenase and PGE2 in synovial cells and dermal fibroblasts45, fragmentation of target-cell DNA into discretely sized pieces46, induction of GM-CSF in normal human lung fibroblasts48, stimulation of complement component C3 in human hepatoma cells49, and depression of cytochrome P450 and drug metabolizing enzymes (ethoxycoumarin deethylase and arylhydrocarbon hydroxylase) in mouse liver50.
The true physiologic role of TNFs in vivo requires knowledge of their relationship to other monokines and lymphokines. In recent years it has become increasingly evident that cytokines acting locally, such as interleukins, interferons, etc., play an important and interdependent role in modifying biological responses. In the immune response, these mediators produce autocrine as well as paracrine effects during T and B cell activation51. For instance, macrophages secrete IL-1, which induces T cells to secrete IL-2 and this in turn causes secretion of IFN-xcex3, which can cause the production of TNF-xcex1 and TNF-xcex2. Human peripheral blood mononuclear cells (PBMC) were shown to be induced by rIL-2 to secrete both TNF-xcex252,53 and TNF-xcex153, and the effect of IL-2 was augmented by rIFN-xcex352,53. In some instances IFN-xcex3 by itself also induced TNF-xcex1/TNFxcex2 production54. TNF-xcex1 production could be seen within 3 hours after induction, reaching peak levels at 48 hr and declining thereafter; TNF-xcex2 production started at a slower rate, requiring greater than 8 hours and reached a peak in 72 hr; IFN-xcex3 did not alter the kinetics of TNF-xcex1/TNF-xcex2 induction by IL-256. Conversely, TNF itself was reported to have stimulated lymphocytes to secrete IFN-xcex354, FS-4 fibroblasts to synthesize IFN-xcex2223, and endothelial cells30, monocytes55,56, and macrophages44 to release Il-1.
Besides regulation of TNF-xcex1/TNFxcex2 production by other cytokines, in order to fully comprehend the true physiological role of TNFs, one must also consider their relationship to the monokine IL-1 and the lymphokine, IFN-xcex3. There are many simiilarities between the biological action of TNFs and IL-1, and the presence of IFN-xcex3 together with TNFs, results in markedly synergistic or antagonistic responses. These actions are briefly described below.
TNFs share a number of biological activities with IL-1, another distinct 17,000 Da protein produced by monocytes, which plays a major role in mediating the immune response58. Some common biological activities of these two monokines include: endogenous pyrogenic activity in vivo56, induction of procoagulant activity in endothelial cells33,38, stimulation of bone resorption and cartilage resorption, stimulation of collagenase and PGE2 production in dermal fibroblasts45,57, suppression of lipoprotein lipase activity in adipocytes, stimulation of growth of fibroblasts58, cytocidal activity against several neoplastic cell lines and anti-tumor activity in vivo. However, differences in biological activity between TNFs and IL-1 also exist, for example, TNF has a rapid direct cytostatic and cytotoxic activity against a wide variety of neoplastic tissues, whereas the cytostatic properties of IL-1 requires coincubation for at least 48 hours and has been observed only against a single human melanoma cell line (A-375). IL-1 also has in vitro adjuvant activity for increasing specific cytotoxic T lymphocytes following coculture of xe2x80x9cnaivexe2x80x9d lymphocytes with allogeneic stimulator cells, an in vitro property not shared with TNF. IL-1 does not activate PMN67. IL-1 does not inhibit stem cell colony formation68.
Several investigators have found that the in vitro tumoricidal activity of TNFs is significantly augmented by co-incubation with IFNs. IFN-xcex1, -xcex2 or -xcex3, while not showing any antiproliferative effects by themselves, synergistically enhanced the cytotoxicity of TNF-xcex1/TNF-xcex2. Synergism between IFN-xcex3 and TNF-xcex2 has also been reported42 for inhibition of hematopoietic cell differentiation. Furthermore, the synergistic effect of IFN-xcex3 appears to be correlated with its ability to induce synthesis of TNF receptors in target cells. It is unclear whether the synergism is solely explained by increased receptor number, or whether other proteins involved in the mechanism of cytotoxicity are also induced by IFN-xcex3.
It is clear from the foregoing discussion that expectations of the in vivo mechanism of action of TNFs are complicated by virtue of their diverse biological effects, their interaction with other monokines and lymphokines, and the target tissues or cells involved.
The objects of this invention are accomplished by a method which comprises administering to an animal (a) a substance against which it is desired to raise an immune response and (b) an adjuvant effective amount of tumor necrosis factor-xcex1 or tumor necrosis factor-xcex2.